The present invention is directed to, among other things, the miniaturization of analytical techniques from a full lab or bench top scale to a lab-on-a-chip (LOAC) device which promises to improve portability, power consumption, reagent and waste volumes, automation, and analysis time. LOAC devices show promise for revolutionizing analysis needed in medical diagnosis, environmental field studies, genomic studies, and for performing synthetic reactions. One of the aspects of LOAC applications is the microfluidics, i.e., the manipulation of small amounts of fluid within the device.
In magnetohydrodynamics (MHD), the magnetohydrodynamic force, FB (N·m−3), which results in fluid flow, is generated by ionic current (normalized to the cross sectional area through which it passes), j (C·s−1·m−2), and a magnetic field, B (T) that is perpendicular to the ionic current vector, j. The magnitude and direction of FB follows the right-hand rule according to the cross-product relationship, FB=j×B.
One of the first approaches that used MHD to pump solutions for microfluidics used direct current (DC) applied to electrodes on either side of a channel filled with electrolyte in the presence of a uniform magnetic field generated from a permanent magnet. This method, when applied to aqueous solutions corroded electrodes and produced bubbles from the electrolysis of water, which interrupted fluid flow.
Two methods have been used to alleviate the problem of water electrolysis and electrode degradation during MHD microfluidics: (1) the addition of redox species to solution, and (2) application of a sinusoidal potential or current waveform to the electrodes.
In the first method, the addition of redox species to the solution allowed pumping at low voltages while keeping the current high to achieve high fluid velocities. The maximum achievable current was proportional to the concentration of redox species; therefore, the higher the concentration, the higher the fluid velocities. However, the introduction of redox species raises concerns about the risk of contamination and interference with analyte detection and with the biocompatibility of the pumping system. Low concentrations of redox species have been shown to be compatible with heart tissue and with alkaline phosphatase, an enzyme commonly used in immunoassay applications. However, the use of low concentrations limits the highest possible velocities that can be achieved.
In the second method, bubble generation was minimized by application of a sinusoidal potential or current waveform at the electrodes while simultaneously altering the magnetic field direction. This approach is called AC-MHD. In one AC-MHD study, a sinusoidal electric current of a frequency greater than 1 kHz was passed through an electrolytic solution to prevent bubble generation and electrode degradation. Higher currents, and therefore higher velocities, were possible at the higher frequencies before bubble formation became a problem, but the magnetic field dropped significantly at frequencies above 1 kHz.
Thus, there is a need for a microfluidic system that is able to pump fluid between two locations, easily reverse fluid flow direction, adjust fluid flow velocity, trap species within a certain volume, mix solutions of different composition, and split off a fluid volume for further handling.
In other aspects, the present invention is directed to, among other things, improving traditional methods for neutrophil counting and three-part differentials (counting granulocyte, lymphocyte, and monocyte sub-populations of leukocytes) that typically rely on automated methods which use blood drawn via venipuncture. Three-part differential tests are essential in monitoring leukopenia in patients receiving chemotherapy. Chemotherapy typically leaves the patient myelosuppressed and susceptible to treatment-induced infection. The differential test is also used to monitor the body's response to latent infections and predict other potential hematopoietic disorders. A point-of-care (POC) hematology device is desirable to provide differential counts to improve the speed at which results are delivered with the same or improved accuracy of traditional flow cytometry or Coulter counting methods, while greatly reducing cost.
A challenge for oncologists and chemotherapy patients is treatment-induced myelosuppression. Monitoring and diagnosing this effect requires multiple draws by painful venipuncture and expensive non-portable hematology analyzers; which themselves require multiple reagents for analysis and a trained lab technician for operation. These limitations restrict the ability to diagnose and monitor myelosuppression at the point-of-care and in low-resource settings.
Since POC systems provide rapid assessment of easily obtained biological samples, such as blood, they are ideally suited for low-resource settings. Although these approaches have many benefits, additional reductions in cost are necessary since many POC diagnostic systems rely on reagents that are difficult and expensive to produce, store and package effectively.
Optical imaging techniques, such as optofluidics, the combination of microfluidics technology and optics, and computer-aided diagnostics also have great promise to reduce the cost of individual screening tests.
Proflavine, an acridine-derived dye, is a small molecule with a high quantum yield (˜35%) of fluorescence. It has previously been used in numerous imaging studies of intact tissue. Proflavine is able to cross cell membranes and preferentially intercalate DNA; more notably, it poorly stains other intracellular structures. This preferential intranuclear staining mechanism makes it an attractive dye for a point-of-care three-part differential due to the fact that leukocytes are the only nucleated cells in whole blood. This unique quality eliminates the need to lyse or remove the red blood cells, as other extant methods require. The dye may be applied to whole blood samples without the need for special environmental controls or lengthy incubation steps, buffers, detergents, or ligand-targeting moieties. This makes it ideal for a point-of-care and avoids long processing times.
In yet other aspects, the present invention is directed to, among other things, addressing the need for high-throughput cell characterization systems capable of morphological characterization of large numbers of living cells in a diverse range of environments, from in vitro cell culture to agricultural applications to biological specimens. There is significant interest in sensing molecular, metabolic, and morphological changes between different cell populations present in a sample or in response to chemotherapeutic interventions; These research areas may encompass basic cell biology, tumorigenesis, drug discovery, and a broad array of other disciplines. Conventional flow cytometry systems require significant investment and have limited portability and are generally limited to exogenous targeting of cellular proteins, requiring a priori knowledge of the target of interest. Optofluidics devices, particularly those coupled to smartphones, have demonstrated excellent portability and show great promise for point of care diagnostic use, but still require the use of pressure-driven bulky syringe pumps, translation stages, and other methods for specimen handling.
Vital clinical applications, such as detection of extremely rare cells in heterogeneous samples, such as circulating tumor cells or cells with intracellular parasites, such as in malaria, make conventional microscopy of a small number of cells unreliable due to under-sampling bias and the need to screen vast numbers of high power fields. Conventional flow cytometry methods are able to screen large numbers of cells but are generally insensitive to intrinsic morphologic or metabolic changes within individual cells, in addition to the lack of portability of these devices.
Commercial imaging cytometry systems currently exist, primarily for research based applications, although some automated methods are available in hematopathology departments in tertiary care centers in the United States. These systems typically acquire data on moderately large numbers of cells, up to one 96-well plate over ten minutes in stationary applications or several tens of microliters for cell suspensions. However, like flow cytometry systems, these are typically limited in scope and insensitive to endogenous reporters of metabolism, such as intrinsic nicotinamide adenine dinucleotide (NADH) or flavin adenine dinucleotide (FAD) fluorescence. Furthermore, due to conventional imaging approaches utilizing complex scanning mirrors and photomultiplier tubes, these systems are generally unsuitable for low-power, and portable operation.
In still further aspects, the present invention is directed to, among other things, addressing ways to scan a sample containing cells. One way is to stain cells on a slide and move the slide beneath the viewing device. This is not an automated approach and requires several steps and skill to handle the sample and perform the staining. A more automated approach is to program the transfer of a sample through the use of microfluidics. Mechanical and electrokinetic pumping are possible options to perform this function.
Electrokinetic pumping has a flat profile that avoids the need to compensate for varied fluid flow across a horizontal plane but is restricted to narrow channels and is highly dependent on the physicochemical properties of the sidewalls (fluid velocity will change depending on the solution properties). Mechanical pumping, such as the use of syringe pumps, requires equipment exterior to the viewing device, moving parts, adds bulk and channels to direct fluid flow, and produces a non-uniform, parabolic flow profile.
Magnetohydrodynamic (MHD) fluid transport is a unique pumping approach that is compatible with a broad range of device shapes and dimensions, does not require moving parts, and provides highly tunable flow patterns and speeds without valves. This pumping approach downsizes, simplifies, and extends the function of the viewing method. MHD offers the flexibility of bidirectional pumping as well as pumping in a circular path. Notably, the entire fluidic manipulation occurs within a microfluidic chip, without necessitating the use of valves and external micropumps. MHD is also compatible with both aqueous and non-aqueous solutions which allows this technique to perform in synthetic organic and biological applications.
As set forth below, the embodiments of the present invention overcome the above described shortcomings in the prior art and provide other advantages.